While there is a large amount of evidence implicating microorganisms in the various forms of periodontal disease, little information exists regarding the role of specific bacterial components as virulence factors in the disease process. Among the large number of prokaryotic species found in a periodontal pocket, four had been more frequently found associated with disease; these are: Bacteroides, Eikenella, Actinobacillus, and Capnocytophaga. Whole cell inoculation of these genera into gnotobionts results in alveolar bone loss. Several lipopolysaccharides (LPS), peptidoglycans (pg) and outer membranes (om) of these genera possess a variable ability to induce alveolar bone resorption in culture, as well as mitogenic potential. Why one LPS or pg or whole om is cytotoxic, or induces alveolar bone loss and why another is either low or devoid of activity is unknown. Which are the active chemical constituents of these molecules is also unknown. This continuation proposal focuses on three of the aspects of host-associated prokaryotes which are associated with virulence: the first is to compare the chemistry of the LPS and pg from these selected Gram-negative periodontopathogens by analysis of their component subunits; secondly, the effects of these molecules and subunits on bone resorption and formation, as well as their ability to activate specific tissue destructive lysosomal hydrolases in human mononuclear phagocytes (MP) and in gingival fibroblast cell lines will be determined. Two isolation protocols will be used for the cell component, so as to compare them chemically and, most importantly, biologically. Controlled fractionation of the LPS and pg will permit the identification of the portions of these molecules which function as virulence factors, and which are relevant to the pathogenesis of periodontal disease and other inflammatory diseases. Thirdly, this grant period will determine the effects of microbial growth physiology on the synthesis and elaboration of extracellular products, including om, hydrolases, proteases, and glycoproteins. The effects of complex and synthetic media on end-products of metabolism, the chemical construction and virulence potential of the LPS and pg, and the synthesis of the hydrolytic enzymes will be studied. Immunoelectron microscopy of ferritin- and gold probes will establish the exact location of the tissue destructive enzymes, collagenase and aminopeptidase.